Targeted modified gulfweed exosome as well as preparation method and application thereof

Abstract

The invention belongs to the field of plant exosome medicines, and particularly discloses a targeted modified gulfweed exosome as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting a natural gulfweed exosome by adopting a conventional technology, carrying out gradient purification on sucrose, then carrying out resuspension by using a PBS buffer solution, carrying out modification by using N, N '-diisopropylcarbodiimide and betulinic acid, then adding urotropine and ceramide for reaction modification, carrying out centrifugation, and then carrying out resuspension by using the PBS buffer solution. The targeting property of the gulfweed exosome to inflammatory tissues / cells is improved through specific modification of various chemical substances, and the utilization rate, the stability and the anti-inflammatory activity of the gulfweed exosome are remarkably improved. Experiments prove that the prepared exosome can significantly reduce the DAI index, increase the colon length and reduce inflammatory cytokines, and the purpose of treating ulcerative colitis is achieved. The invention provides a new modification method for improving the targeting property and activity of the plant exosome, and provides a new treatment approach for inflammatory intestinal diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant exosome medicine, and particularly relates to a targeted modified horsetail exosome, its preparation method and application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of nonspecific chronic inflammatory diseases of the gastrointestinal tract with unknown causes, including ulcerative colitis (UC), Crohn's disease (CD), and indeterminate colitis (IC). UC is an inflammatory process limited to the colonic mucosa and submucosa, most commonly affecting the sigmoid colon and rectum. Early inflammation may involve macrophage infiltration into the intestinal mucosal stroma, disrupting the intestinal mucosal barrier and leading to immune dysregulation, thus triggering inflammation. Symptoms include weight loss, diarrhea, bloody stools, fever, impaired gastric motility, and shortened colon. CD is characterized by transmural inflammation and skip lesions; transmural inflammation can lead to intestinal wall fibrosis and narrowing, as well as sinus tract formation, causing perforation and intestinal fistula. IBD differs significantly from ordinary enteritis. Ordinary enteritis is generally an acute inflammation that can be cured with anti-infective treatment. However, IBD has a longer course, recurs frequently, and patients experience fibrosis and hardening of the intestinal wall, leading to progressively worsening intestinal function. This severely impacts the patient's quality of life, and its treatment is difficult to standardize, resulting in high disease care costs. Currently, it is extremely difficult to cure.

[0003] Currently, drug therapy is the primary treatment for IBD. For example, ulcerative colitis (UC) treatment utilizes various medications including aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics. Aminosalicylic acid preparations are the first-line drugs for mild to moderate ulcerative colitis, but side effects may include nausea, vomiting, and headache. Glucocorticoids are suitable for moderate to severe ulcerative colitis, but long-term use may lead to side effects such as osteoporosis, hypertension, and diabetes. Immunosuppressants and biologics can reduce the use of glucocorticoids, but may increase the risk of infection. Therefore, researching and developing safe and reliable drugs for treating inflammatory bowel disease is of great significance for alleviating patient suffering and improving their quality of life.

[0004] Exosomes, as natural nanocarriers secreted by cells, have received widespread attention and research. Compared with artificially synthesized nanocarriers, exosomes have unique advantages in the field of drug delivery. The components of exosomes are all derived from cells, avoiding the cytotoxicity and biocompatibility problems associated with the use of artificially synthesized liposomes, polymers, and nanosilicon nanomaterials. Because exosomes inherit phospholipids and surface proteins from cells, their specific cellular components make their functions complex and diverse, and they possess a certain degree of targeting. They can participate in physiological processes such as immune responses, antigen presentation, cell migration, cell differentiation, and tumor invasion, regulating intercellular communication and improving drug delivery efficiency, enabling specific drug delivery, cross-biological barrier delivery, and immune immunity. The proteins and genes carried by exosomes themselves have cellular regulatory capabilities and can play a therapeutic role in specific diseases.

[0005] Plant-derived exosomes possess a phospholipid bilayer structure similar to that of animal-derived exosomes, carrying abundant proteins, lipids, nucleic acids, and plant-specific secondary metabolites, and have been proven to have good biocompatibility and oral safety. However, natural plant exosomes themselves have low targeting specificity and are also taken up by normal tissues and organs during their action, resulting in low utilization. To increase the accumulation of plant exosomes in specific tissues and organs, researchers have modified the surface of natural exosomes. Numerous studies have shown that exosome surface modification can enhance their targeting and enable drug delivery to specific tissues or organs for treatment. Animal exosomes can express various disease-related proteins through genetic engineering, while plant exosomes are difficult to modify. Currently common modification methods, such as liposome modification, often lead to changes in the morphology and activity of plant exosomes. There is still a need to find a safe and rapid modification method to specifically modify plant exosomes to improve their targeting and activity, thereby enhancing therapeutic effects.

[0006] Horsetail grass is an annual herb belonging to the genus Setaria of the Poaceae family. According to records in *Compendium of Materia Medica* and *Zhejiang Medicinal Plants*, horsetail grass has the effects of clearing heat and dampness, dispelling wind and improving eyesight, detoxifying, and killing parasites. It is mainly used to treat wind-heat colds, jaundice, infantile malnutrition, dysentery, painful urination, red and painful eyes, red and swollen eyes, carbuncles, common warts, and sores. Horsetail grass is rich in silicic acid, which can help the body absorb calcium and nourish nails, skin, hair, bones, and connective tissue throughout the body; it is rich in chromium, flavonoids, saponins, and astringents, thus preventing arteriosclerosis; it can also help remove excess oil from the skin and hair, making hair grow healthier, thicker, and more elastic; in addition, horsetail grass can also be used to stop bleeding wounds and promote healing. Modern pharmacological research mainly focuses on alkaloid active ingredients. Horsetail grass mainly contains a variety of benzylisoquinoline alkaloids, including berberine, thalidomide, hainan terpinene, palmatine, herba alkaloid, magnoflorine, and thalidomide, but there are currently no reports of horsetail grass-derived exosomes. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a targeted modified *Pteris vittata* exosome, its preparation method, and its applications. This invention achieves targeted transport by modifying the surface of *Pteris vittata* exosomes, significantly improving their utilization rate, stability, and anti-inflammatory activity. It has great potential for application in the preparation of drugs for treating intestinal diseases, especially inflammatory bowel diseases such as enteritis, ulcerative colitis, and ulcerative colitis.

[0008] The primary objective of this invention is to provide a method for preparing targeted modified horsetail exosomes.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for preparing targeted modified horsetail exosomes includes the following steps:

[0011] S1. Take fresh horsetail grass, wash it, dry it and crush it for later use. Add PBS buffer and put it into a homogenizer to make a homogenate. The homogenate is centrifuged multiple times to remove impurities and obtain supernatant. After ultracentrifugation of the supernatant, a precipitate containing horsetail grass exosomes is obtained. The precipitate is resuspended in pre-cooled PBS solution and purified further using the sucrose density gradient method to obtain horsetail grass exosomes.

[0012] S2. Resuspend the horsetail exosomes obtained in step S1 in pre-cooled PBS buffer, add N,N'-diisopropylcarbodiimide and betulinic acid, mix thoroughly and react, then add hexamethylenetetramine and ceramide, centrifuge after reaction, and resuspend the precipitate in PBS buffer to obtain the targeted modified horsetail exosomes.

[0013] Preferably, the mass ratio of horsetail grass to buffer solution in step S1 is 1:1-3.

[0014] Preferably, the differential centrifugation in step S1 is performed by centrifuging at 2000g for 20 minutes, at 10000g for 1 hour, and at 40000g for 1 hour in sequence; the ultracentrifugation is performed by ultracentrifugation at 120000g for 2 hours at 4°C.

[0015] Preferably, the specific operation of the sucrose density gradient purification method in step S1 is as follows: the precipitate suspension is placed in a centrifuge tube, and sucrose solutions with mass concentrations of 15%, 30%, 45%, and 60% are added respectively. After centrifugation at 120,000g for 2 hours, the intermediate sample bands of 30% and 45% are collected, diluted with pre-cooled PBS, and then centrifuged at 120,000g for 2 hours to remove sucrose.

[0016] Preferably, the concentration of the *Pterocarya stenoptera* exosome suspension in step S2 is 0.5-1 mg / mL, and the mass ratio of betulinic acid to *Pterocarya stenoptera* exosomes is 1:0.8-1.

[0017] Preferably, the amounts of N,N'-diisopropylcarbodiimide, betulinic acid, hexamethylenetetramine, and ceramide added in step S2 are in an equimolar ratio.

[0018] Plant exosomes, as vesicles enclosed in a lipid bilayer, have a complex and diverse chemical composition. The glycolipids and glycoproteins on the exosome membrane contain abundant hydroxyl groups (-OH), and the proteins and lipids they carry naturally contain carboxyl groups (-COOH) and amino groups (-NH2). These functional groups are the basic structural units that constitute biological macromolecules such as proteins, lipids, nucleic acids, and carbohydrates.

[0019] This invention utilizes compounds with similar structures, such as N,N'-diisopropylcarbodiimide (DIC), to activate the carboxyl groups in *Phyllostachys edulis* exosome protein molecules, forming an active lipid intermediate. This intermediate further undergoes an amidation reaction with the amino groups in the protein molecule to form peptide bonds, which are used to modify exosome proteins, improving drug delivery efficiency and targeting. Simultaneously, betulinic acid, a compound with diverse biological activities but relatively weak anti-inflammatory activity, can react with the amino terminus of *Phyllostachys edulis* exosome proteins. This reaction typically occurs via covalent bonding; betulinic acid can form an ester bond with the amino terminus of the exosome protein through its carboxyl group, thereby covalently linking betulinic acid to the exosome protein for functionalization.

[0020] On the other hand, this study found that betulinic acid may have a certain affinity for inflammatory cells, enhancing the targeting of modified *Pteris vittata* exosomes to inflammatory cells / tissues. The modified *Pteris vittata* exosomes specifically act on inflammatory cells such as macrophages and neutrophils, making inflammatory cells / tissues key targets, while exhibiting low cytotoxicity to normal cells. In experiments, they effectively inhibited the secretion of pro-inflammatory factors, suppressing excessive activation of inflammatory cells by reducing the activity of inflammatory signaling pathways.

[0021] Urotropin has a cyclic structure and contains multiple amino and imino groups in its molecule, exhibiting unique adsorption properties. Ceramides have certain adjuvant anti-inflammatory effects, mainly through mechanisms such as repairing the skin barrier and regulating immune responses. This invention utilizes the interaction between urotropin and ceramides to adsorb and distribute urotropin within the cross-linked structure on the surface of *Pteris vittata* exosomes, inducing their migration to target inflammatory cells / sites. The mechanism is likely due to the complexation of urotropin with the hydroxyl groups on the exosome surface. The amino groups in urotropin act as ligands, forming complexes with the hydroxyl groups on the *Pteris vittata* exosome surface. This complexation reaction is mainly based on hydrogen bonding between amino and hydroxyl groups, increasing the stability and functional activity of exosomes. This allows exosomes to better carry and deliver drugs to target cells or organs, thereby improving therapeutic efficacy.

[0022] Another object of the present invention is to provide a plant exosome that has been targeted and modified by the above method.

[0023] The present invention also provides the application of the above-mentioned targeted modified plant exosomes in the preparation of drugs for treating intestinal diseases, including inflammatory bowel diseases such as enteritis, ulcerative colitis, and ulcerative colitis.

[0024] Another aspect of the present invention provides a medicament for treating inflammatory bowel disease, the active ingredient mainly comprising the above-mentioned targeted modified horsetail exosomes, and medically acceptable excipients or carriers, wherein the excipients include one or more of physiological saline, phosphate buffer, DMSO, vegetable oil, etc., and are prepared into a medically acceptable dosage form for treatment.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention provides a targeted modified *Phyllostachys edulis* exosome, its preparation method, and its applications. Specific modification of various chemical substances enhances the targeting of *Phyllostachys edulis* exosomes to inflamed tissues / cells, further improving utilization and stability. Experiments demonstrate that the modified *Phyllostachys edulis* exosomes exhibit high safety and significantly enhance in vitro and in vivo anti-inflammatory activity without affecting normal cells. Animal model experiments with ulcerative colitis show that the modified *Phyllostachys edulis* exosomes can significantly reduce hematochezia and loose stool index, increase colon length, and inhibit inflammatory factors, achieving effective treatment for ulcerative colitis. Furthermore, it is more effective than unmodified exosomes in the treatment of inflammatory bowel disease. This invention provides a novel modification method for improving the targeting and activity of plant exosomes, offering a new approach to the prevention and treatment of inflammatory bowel diseases. Attached Figure Description

[0027] Figure 1 The images show electron micrographs of the horsetail exosomes prepared in this invention before and after modification.

[0028] Figure 2 The image shows the nanoparticle size distribution of the *Pterocarya stenoptera* exosomes prepared in this invention before and after modification. A represents unmodified *Pterocarya stenoptera* exosomes, and B represents modified *Pterocarya stenoptera* exosomes 2.

[0029] Figure 3 The images show the stained electron microscope images of macrophages before and after modification of the horsetail exosomes prepared in this invention.

[0030] Figure 4 The illustration shows the results of the in vitro anti-inflammatory effect analysis of the horsetail exosomes prepared in this invention before and after modification.

[0031] Figure 5 The results show the effect of horsetail exosomes prepared in this invention on colon length in ulcerative colitis model mice, compared with the normal group. ## P < 0.01; compared with the model group, *P < 0.05, **P < 0.01.

[0032] Figure 6 The effects of horsetail exosomes prepared in this invention on the expression of inflammatory cytokines in the colon tissue of a mouse model of ulcerative colitis were compared with those in the normal group. && P < 0.01; compared with the model group, *P < 0.05, **P < 0.01.

[0033] Figure 7 The figure shows the effect of the horsetail exosomes prepared in this invention on the blood routine of model mice.

[0034] Figure 8 To compare the efficacy of the horsetail exosomes prepared in this invention with the control group in treating inflammatory bowel disease, the average weight change of mice in each group from day 1 to day 14 was statistically analyzed (mean ± SD, compared with the control group, *P < 0.05).

[0035] Figure 9 The recovery rate of inflammatory area in mice with inflammatory bowel disease treated with the horsetail exosomes prepared in this invention and the control group between days 7 and 14 (mean ± SD, compared with the control group, *P<0.05, **P<0.01, ***P<0.001). Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0038] Example 1: A method for preparing targeted modified horsetail exosomes

[0039] (1) Extraction of horsetail grass exosomes: Fresh horsetail grass was cleaned, dried at 50℃, and pulverized to 100 mesh for later use. PBS buffer was added to the powder at a mass ratio of 1:1, and homogenized in a homogenizer. The homogenate was then transferred to a centrifuge tube. The tube was centrifuged at 2000g for 20 min, at 10000g for 1 h, and at 40000g for 1 h. Impurities were removed by differential centrifugation, and the precipitate was discarded to obtain the supernatant. The supernatant was then subjected to ultra-high speed centrifugation at 120000g for 2 h at 4℃ to obtain a precipitate containing horsetail grass exosomes. The precipitate was resuspended in pre-cooled PBS solution to obtain a precipitate suspension.

[0040] (2) Place the precipitate suspension in a centrifuge tube and add sucrose solutions with mass concentrations of 15%, 30%, 45%, and 60%, respectively. After centrifuging at 120,000g for 2 hours, collect the intermediate sample bands of 30% and 45%. Add pre-cooled PBS to dilute and centrifuge at 120,000g for 2 hours to remove sucrose and obtain the precipitate. Resuspend the precipitate with pre-cooled PBS buffer and adjust the content of horsetail exosomes to 1 mg / mL for later use (extracted natural horsetail exosomes).

[0041] (3) Take 1 mL of horsetail exosomes and add an alcohol solution containing 0.28 mg N,N'-diisopropylcarbodiimide (DIC) and 1 mg betulinic acid (use a small amount of ethanol to dissolve N,N'-diisopropylcarbodiimide and betulinic acid). Mix thoroughly and react at room temperature for 12 h. Centrifuge at 120,000 g for 2 h. Resuspend the precipitate (modified horsetail exosome 1) in 1 mL of pre-cooled PBS buffer. Add 0.26 mg urotropine (HMTA) and 1.18 mg ceramide (CERAMIDE). Mix thoroughly and react at room temperature for 12 h. Centrifuge at 120,000 g for 2 h. Resuspend the precipitate in 1 mL of PBS buffer to obtain the targeted modified horsetail exosomes (modified horsetail exosome 2).

[0042] The morphology and structure of *Pteris vittata* exosomes before and after modification were observed using transmission electron microscopy, and the particle size of *Pteris vittata* exosomes before and after modification was determined by dynamic light scattering (DLS).

[0043] Figure 1Electron microscopy revealed that the exosomes of *Pteris vittata* contain a typical lipid bilayer membrane structure and are flattened spherical vesicles, consistent with the characteristics of plant exosomes. Furthermore, there was no significant difference in morphology and structure before and after modification. Figure 2 DLS testing revealed that the exosomes of *Pteris vittata* before and after modification were mainly concentrated at approximately 140 nm and 160 nm, exhibiting a unimodal normal distribution. The polydispersity indices were 0.153 and 0.168, respectively, indicating good particle dispersibility. Furthermore, there was no significant change in particle size before and after modification.

[0044] Example 2: Stability and targeting of horsetail exosomes

[0045] The natural horsetail exosomes and modified horsetail exosomes 1 and 2 were extracted and placed in simulated gastric juice and incubated in a water bath at 37°C for 2 hours. After 2 hours, the exosomes were collected by centrifugation, washed 3 times, and then the exosomes were tested to see if they still had the original biological morphology / function.

[0046] The results showed that after exposure to simulated gastric juice at 37°C for 2 hours, the modified *Pterocarya stenoptera* exosome 2 exhibited no significant change in particle size, demonstrating good stability. In contrast, the membrane structure of both the unmodified and modified *Pterocarya stenoptera* exosome 1 was slightly damaged, potentially affecting their biological functions. This may be because the modified *Pterocarya stenoptera* exosome 2 utilizes the amino group in urotropine as a ligand to form a complex with the hydroxyl group on the exosome surface. This complexation reaction is primarily based on hydrogen bonding between the amino and hydroxyl groups, increasing the stability and functional activity of the exosomes.

[0047] The *Pterocarya stenoptera* exosomes extracted in Example 1 and the modified *Pterocarya stenoptera* exosomes were fluorescently labeled (PKH67). RAW264.7 cells were revived, and after reaching approximately 50%-60% confluence, they were digested, counted, and seeded into well plates. After seeding, the cells were incubated at 37°C with 5% CO2 for 24 hours. Cells were then treated with the fluorescently labeled *Pterocarya stenoptera* exosomes and the modified *Pterocarya stenoptera* exosomes, respectively, for 18 hours, fixed with 4% paraformaldehyde solution, and observed under a fluorescence microscope.

[0048] The results are as follows Figure 3 As shown, after treating macrophages Raw264.7 with 0.1 mg / mL horsetail exosomes and modified horsetail exosomes for 18 hours, the proportion of targeted cells in the modified horsetail exosome treatment group was significantly increased, which was about 30 times higher than that in the group treated with extracted natural horsetail exosomes.

[0049] Example 3: In vitro anti-inflammatory effect of horsetail exosomes

[0050] The in vitro anti-inflammatory effects of the natural horsetail exosomes and modified horsetail exosomes prepared in Example 1 were determined. Specifically, an LPS-induced macrophage cytokine inflammation model was used. Natural horsetail exosomes, modified horsetail exosome 1, and modified horsetail exosome 2 were added and cultured for 24 h. After collecting the cells, RNA was extracted, and the mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α were detected by qRI.

[0051] The results are as follows Figure 4 As shown, the mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α were increased in both the model group and the control group, indicating successful model establishment. Compared with the model group, the mRNA levels of LPS-induced inflammatory factors IL-1β, IL-6, and TNF-α were reduced in all three groups: Example 1 (natural horsetail exosomes), Example 2 (modified horsetail exosome 1), and Example 3 (modified horsetail exosome 2). The modified horsetail exosome 2 group showed the most significant anti-inflammatory effect.

[0052] Example 4: Study on the effect of horsetail exosomes on ulcerative colitis

[0053] 1. Materials and Methods

[0054] 1.1 Construction of an animal model of ulcerative colitis

[0055] C57BL / 6J mice aged 6-8 weeks, weighing 18-22g, were randomly divided into four groups: a normal group, a model group, Example 1 group (natural horsetail exosomes), Example 2 group (modified horsetail exosome 1), and Example 3 group (modified horsetail exosome 2), with 10 mice in each group. During the first week, all groups of mice were acclimatized in a standard animal room (25±1℃) and provided with standard food and drinking water. During the second week, the normal group mice had free access to water, while the other groups were given 3% DSS drinking water for 7 consecutive days to establish the ulcerative colitis mouse model. The drinking water was changed daily.

[0056] 1.2 Dosing regimen

[0057] In Examples 1-3, mice were administered 10 mg / kg of the corresponding exosomes by gavage once daily.

[0058] Mice in the normal group and the model group were administered an equal volume of physiological saline by gavage once daily.

[0059] The above medication was administered via gavage for 14 consecutive days.

[0060] 1.3 Disease Activity Index Assessment

[0061] From the first day of modeling, the weight, stool characteristics and bloody stool of each group of mice were observed daily. Records and scores were made after successful modeling and treatment. The scoring criteria based on the Disease Activity Index (DAI) are shown in Table 1.

[0062] Table 1

[0063]

[0064]

[0065] 1.4 Colon length

[0066] Mice in each group were dissected, their colons were separated, and the length of the colons was recorded.

[0067] 1.5 Detection of the expression of inflammatory cytokines in colon tissue

[0068] A portion of colon tissue was placed in a centrifuge tube containing RNA preservation solution and stored at -80℃ for RT-qRCR to detect the expression levels of IL-17A, IFN-γ, and TNF-α. The specific steps were performed according to the kit instructions.

[0069] 1.6 Safety evaluation of horsetail exosomes

[0070] Healthy mice were injected intravenously with the horsetail exosomes prepared in Example 1 of this invention. Samples were taken 24 hours later for H&E staining and routine blood tests.

[0071] 1.7 Statistical Methods

[0072] Data were processed using SPSS 22.0 software. The obtained data are expressed as mean ± standard deviation (x ± s). Normality test and homogeneity of variance test were performed on each group of data. One-way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant.

[0073] 2. Analysis of Experimental Results

[0074] 2.1 DAI index in mice

[0075] Before modeling, all mice were in good health, with normal, spherical or strip-shaped stools and increased weight. The DAI scores before and after treatment are shown in Table 2. Compared to the normal group, mice in the model group and the example group showed decreased activity, loose stools, diarrhea, and weight loss starting from the third day after drug administration, indicating successful modeling. Two weeks after gavage treatment, mice in the model group showed gross hematochezia, mice in the example 3 group had dry, formed stools, and mice in examples 1 and 2 had soft stools. Compared to the model group, the DAI scores of examples 1-3 were all reduced to varying degrees, with the DAI index of example 3 being significantly lower than that of examples 1 and 2, indicating the best therapeutic effect on the ulcerative colitis mouse model.

[0076] Table 2

[0077]

[0078] 2.2 Effects of horsetail exosomes on colon length in ulcerative colitis model mice

[0079] like Figure 5 As shown, compared with the normal group, the colon length of mice in the model group was significantly shortened. After treatment, compared with the model group, the colon length of groups 1 and 2 increased significantly, and the colon length increase in group 3 was significantly better than that in the other groups.

[0080] 2.3 Effects of *Pteris vittata* exosomes on the levels of inflammatory cytokines in colon tissue of a mouse model of ulcerative colitis

[0081] like Figure 6 As shown, compared with the normal group, the levels of 1L-17A, IFN-γ, and TNF-α in the model group were significantly increased (P<0.01); compared with the model group, the levels of 1L-17A, IFN-γ, and TNF-α in each example group were reduced to varying degrees, with the reduction effect being the most significant in Example 3 group.

[0082] 2.5 Safety evaluation of horsetail exosomes

[0083] H&E staining results of mouse organ sections showed that the morphology and cell morphology of various organs and tissues (heart, liver, lung, spleen, and kidney) were similar to those of normal mice after injection of exosomes. Figure 7 The blood routine results showed that the levels of WBC (white blood cells), PLT (platelets), Neutrophils, Lympocytes, Monocytes, RBCs (red blood cells), and HGB (hemoglobin) in mice injected with exosomes were not significantly different from those in the normal group of mice, and were all within the normal range. Therefore, the targeted modified horsetail exosomes prepared in this invention have high safety.

[0084] Example 5: The therapeutic effect of horsetail exosomes on inflammatory bowel disease

[0085] Eight-week-old male C57BL / 6J mice, weighing 18-22g, were randomly divided into a normal group, a control group (physiological saline), Example 1 group (natural horsetail exosomes), Example 2 group (modified horsetail exosome 1), and Example 3 group (modified horsetail exosome 2), with 10 mice in each group. The mice were acclimatized for 5 days.

[0086] To prepare a trinitrobenzenesulfonic acid (TNBS) solution, dissolve TNBS in a 40-50% ethanol solution at a concentration of 50 mg / mL.

[0087] On the first day of the experiment, mice in both the control and example groups were anesthetized, and TNBS solution at a dose of 100 mg / kg was slowly injected into the rectum via a catheter, with the mice kept in a head-down position for 30-60 seconds to prevent spillage. From days 1 to 3 and from days 8 to 10 of the experiment, the example and control groups were administered 10 mg / kg of the corresponding exosomes and an equal volume of physiological saline rectally daily. On day 7 of the experiment, half of the mice in each group were sacrificed to observe and record changes in body weight and area of ​​inflammation. The other half of the mice were sacrificed on day 14 of the experiment, and changes in body weight and area of ​​inflammation were observed and recorded.

[0088] Results Analysis

[0089] In this embodiment, changes in mouse body weight and the recovery rate of inflamed area were used as indicators to evaluate the treatment efficacy of inflammatory bowel disease. Statistical results are shown below. Figure 8 and Figure 9 The weight change trends of the groups in Examples 1-3 show that, compared with the control group, there is a significant therapeutic effect on inflammatory bowel disease from day 3 to day 14 of the experiment. From day 11 to day 14, the weight changes of the example groups compared with the control group are statistically significant, especially the treatment effect of Example 3 is the most significant. At the same time, the recovery rate of the inflamed area after treatment in Example 3 group is significantly higher than that of other groups.

[0090] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing targeted modified horsetail exosomes, characterized in that, Includes the following steps: S1. Take fresh horsetail grass, wash it, dry it and crush it for later use. Add PBS buffer and put it into a homogenizer to make a homogenate. The homogenate is centrifuged multiple times to remove impurities and obtain supernatant. After ultracentrifugation of the supernatant, a precipitate containing horsetail grass exosomes is obtained. The precipitate is resuspended in pre-cooled PBS solution and purified further using the sucrose density gradient method to obtain horsetail grass exosomes. S2. Resuspend the horsetail exosomes obtained in step S1 in pre-cooled PBS buffer, add N,N'-diisopropylcarbodiimide and betulinic acid, mix thoroughly and react, then add hexamethylenetetramine and ceramide, centrifuge after reaction, and resuspend the precipitate in PBS buffer to obtain the targeted modified horsetail exosomes.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of horsetail grass to buffer solution is 1:1-3.

3. The preparation method according to claim 1, characterized in that, The differential centrifugation in step S1 is to centrifuge at 2000g for 20 min, 10000g for 1 h, and 40000g for 1 h in sequence; the ultracentrifugation is to centrifuge at 120000g for 2 h at 4℃.

4. The preparation method according to claim 1, characterized in that, The specific operation of the sucrose density gradient purification method described in step S1 is as follows: the precipitate suspension is placed in a centrifuge tube, and sucrose solutions with mass concentrations of 15%, 30%, 45%, and 60% are added respectively. After centrifugation at 120,000g for 2 hours, the intermediate sample bands of 30% and 45% are collected. After dilution with pre-cooled PBS, the sucrose is removed by centrifugation at 120,000g for 2 hours.

5. The preparation method according to claim 1, characterized in that, The concentration of the *Pteris vittata* exosome suspension in step S2 is 0.5-1 mg / mL, and the mass ratio of betulinic acid to *Pteris vittata* exosomes is 1:0.8-1.

6. The preparation method according to claim 1, characterized in that, The amounts of N,N'-diisopropylcarbodiimide, betulinic acid, hexamethylenetetramine, and ceramide added in step S2 are in an equimolar ratio.

7. A targeted modified horsetail exosome, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. The use of the targeted modified horsetail exosomes according to claim 7 in the preparation of a drug for treating inflammatory bowel disease.

9. A medicament for treating inflammatory bowel disease, characterized in that, It includes the targeted modified horsetail exosomes as described in claim 7, and medically acceptable excipients or carriers.

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